Compositions and methods for target delivering a bioactive agent to aquatic organisms

ABSTRACT

Biodegradable and nutritionally attractive composition comprising biocidal or antibiotic compounds and/or microbes having bio-adhesion and controlled buoyancy properties are selectively fed to an aquatic organism in open or closed water-bodies, and bioactive components are released upon contact with mucosal tissues such as gill, skin or along the digestive tract of the selected aquatic organism.

This application is the National Phase filing of international patentapplication number PCT/US2013/027095, filed 21 Feb. 2013, and claimspriority of U.S. provisional patent application No. 61/601,290, filed 21Feb. 2012, the entirety of which applications are incorporated byreference herein for all purposes. This work was supported through apurchase order from the U.S. Department of the Interior, U.S. GeologicalSurvey.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to biodegradable and nutritionally attractivecomposition comprising biocidal or antibiotic compounds and/or microbeshaving bio-adhesion and controlled buoyancy properties for selectivelyfed to an aquatic organism in open or close water-bodies, and bioactivecomponents are released upon contact with mucosal tissues such as gill,skin or along the digestive tract of the selected aquatic organism.

2. Description of the Related Art

Non-indigenous aquatic species are rapidly spreading worldwide, causingboth a severe loss of global biodiversity and environmental and economicdamages [1, 2, 3]. In addition to direct effects on habitat quality, theexpected climate changes will foster the expansion of invasive speciesinto new areas and magnify the effects already present by alteringcompetitive dominance, increasing predation and infectious diseases.Aquatic species that are considered invasive are non-native species, asthey are free from natural predators, reproduce rapidly and aggressivelycompete with native species. Invasive predatory species prey upon nativespecies and disrupt their aquatic food web. They can affect propertyvalues, and influence economies of water-dependent communities.

For example, many non-native aquatic plants, animals and microscopicorganisms have been introduced into the Great Lakes since the early1800s, either accidentally or intentionally. Many of them over-populatethe lakes and surrounding rivers. They prey on native fish and plants,and disrupt the ecosystem in the lakes. They also harm the recreationaland agricultural activities by damaging boats and gear, underwatercables, oil rig platforms, buoys, fishing nets, clogging water pipes andhydro-power facilities, jamming the fresh water supply chain and chokingoff irrigation systems in the region. Losses in the U.S. alone areestimated at $78.5 billion annually [4]. Recent efforts across manycountries have highlighted the urgent need for more rigorous andcomprehensive management programs to prevent and contain the worldwidespread of non-indigenous species.

The invasion of Zebra mussel (Dreissena polymorpha) and Asian clam(Corbicula fluminea) are of particular concern given their ability torapidly cover the surface of hard submerged substrates, reducephytoplankton biomass and hence disturb pelagic food webs and act asmajor macro fouling species of water intake structures and pipes used inmunicipal, agricultural, industrial, and power station water systems [5,6]. Asian clams are found in 36 of the contiguous states of the UnitedStates as well as in Hawaii. The zebra mussel, introduced into the U.S.in 1986, has spread rapidly throughout the Great Lakes, St. LawrenceRiver, and waterways associated with the Mississippi River. It isexpected that the mussels will, within 20-25 years, infest most areassouth of Central Canada and north of the Florida Panhandle from thePacific Coast to the Atlantic Coast. As the zebra mussel advances, theprognosis for native freshwater bivalve populations is bleak, especiallyfor those populations of species considered threatened and endangered[7].

Another harmful invader is the round goby Neogobius melanostomus, whichis one of the most wide-ranging invasive fish on earth. The fish hassubstantial introduced populations within the Laurentian Great Lakeswatershed, the Baltic Sea and several major European rivers. Neogobiusmelanostomus inhabit a wide range of temperate freshwater andbrackish-water ecosystems and without establishing rigorous managementprograms will probably continue to spread via ballast water, accidentalbait release and natural dispersal worldwide [8].

There are many methods of controlling the spread of invasive species.These methods include the mechanical removal such as dredging, chaindragging and hand raking, predator removal, and chemical, biochemicaland biological control. It is equally important to manage the invasivespecies in a safe, environmentally responsible and cost effectivemanner. For example, in order to find less harmful methods to controlinvasive mussels, New York State Museum's (NYSM) Field ResearchLaboratory screened more than 700 bacterial isolates as potentialbiological control agents against zebra and quagga mussels. As a result,they found a highly effective and lethal strain isolate of Pseudomonasfluorescens (CL145A) against these mussels (U.S. Pat. No. 6,194,194).This harmless bacterium is present in all North American water bodiesand even in the average household kitchen and refrigerator [9].

Application of biocides and toxicants is one of the effective ways toreduce a population of invasive species. However, application techniqueshave not been perfected and as a result those methods have been quiteineffective in eradicating the invasive organism. Another disadvantageis that many toxicants in use such as sodium hypochlorite, surfactants,ammonium salts, N-triphenylmethyl-morpholine have either low toxicity ornon-selectively affect the entire water ecosystems. For example,Clam-Trol, produced by Betz Chemicals, H130 produced by Calgon Corp. and4-trifluoroethyl-4-nitrophenol marketed as Bayluscide® (Bayer) areacutely toxic to fish and other aquatic organisms and are believed to bequite persistent in the environment [10]. To date, none of thosechemical treatments seems likely to replace simple chlorination as thestandard treatment for zebra mussels.

The two most widely used fish toxicants in aquatic systems are Rotenoneand antimycin A. Rotenone, is a botanical pesticide registered by theEPA for piscicidal (fish kill) uses. The chemical is related toisoflavonoid compounds derived from the roots of Derris spp.,Lonchocarpus spp., and Tephrosia spp., and primarily found in SoutheastAsia, South America, and East Africa, respectively. Rotenone productsare classified as Restricted Use Pesticides (RUP) due to acuteinhalation, acute oral, and aquatic toxicity (see EPA 738-R-07-005,March 2007, Registration Eligibility Decision for Rotenone). Rotenonedoes not dissolve in water. In order to disperse it in water so that itcan be effective at low concentrations, rotenone must be formulated withsolvents. There are a couple of commercial liquid emulsion productscontaining rotenone as the active ingredient that can be used fortreating aquatic systems. For example, one product is calledNusyn-Noxfish®, the other CFT Legumine®. These piscicides are usuallyapplied by spraying the emulsion on the surface of the water. However,these emulsion-type piscicidal compositions have many disadvantages, asdescribed below.

Antimycin A is a relatively new fish toxicant, and primarily applied asa single management tool. Over the past decade antimycin A has been usedby Federal and state agencies to restore threatened/endangered fish totheir native habitats (see EPA 738-R-07-007, May 2005, RegistrationEligibility Decision for Antimycin A). Antimycin A is also a RestrictedUse Pesticide registered by EPA for piscicidal (fish kill) uses. Derivedas a fermentation product from Streptomyces mold, the chemical isapplied directly to water to renovate recreational fish populations andto remove scaled fish from catfish fingerling and food fish productionponds.

This toxicant is marketed under the trade name of “Fintrol.” Currently,there are three registered formulations of antimycin A available.Fintrol-5 consists of antimycin A coated on sand grains in such a way asto release the toxicant evenly in the first 5 feet of water—as the sandsinks; Fintrol-15 which releases it in the first 15 feet of depth, and aliquid, Fintrol Concentrate, which was developed for use in very shallowrunning waters and streams. Since its introduction, antimycin A hasbecome an attractive pesticide because of its relative specificity tofish, i.e., the minimal concentrations that kill fish are consideredharmless to other aquatic life and mammals. It is effective in verysmall concentrations against all life stages of fish, egg through adult.Its respiratory inhibiting properties are irreversible at lethaldosages, and as importantly, it rapidly degrades in open environment.

Efforts to better control the release of the toxicant are well known,particularly in the agricultural industry. For example, U.S. Pat. Nos.3,851,053 and 4,400,374 disclose various polymers with extendeddiffusion path length. Typically, agents incorporated are organicpesticides, and the matrix type is an elastomer such as natural rubber,styrene-butyl styrene rubber, and the like. It is, however, well knownin the art that almost all organic and inorganic pesticidal agents lacksolubility in those plastic matrices.

Other known encapsulating systems include; U.S. Pat. Nos. 3,059,379 and4,428,457 in which a core-granulated fertilizer is encapsulated inporous thin film; U.S. Pat. No. 4,019,890 in which granular fertilizersis coated with a water-resisting layer forming a jelly-like gel coating.U.S. Pat. No. 2,891,355 relates to coating foamed polystyrene particleswith a solution of fertilizers and nutrients, adding water, and pottinga plant therein. Further, Villamar et al. [11] describes the preparationof complex microcapsules (CXMs) consisting of dietary ingredients andlipid-wall microcapsules (LWMs) embedded in particles of a gelledmixture of alginate and gelatin to obtain a single food-particle typeused to provide suspension feeders with dietary nutrients. Otherfertilizers such as urea can be coated in a granular form as taught inU.S. Pat. No. 3,336,155, thus retarding solution in ground waters. U.S.Pat. No. 3,276,857 teaches that a fertilizer can be encapsulated withasphalt or various waxes and, thus, emission into the environment isslowed. However, none of this prior art discloses a particle wherein theactive agent remains within an intact particle even after exposure inwater and wherein it is being released only after consumption by anorganism. One approach to deliver a toxicant directly to the invasivespecies is through conventional aquatic feeds in a dry, semi or wet softform as a pelleted or flaked feed. These feeds however, rapidlydeteriorate in water, with physical decomposition and breakdown of thefeed starting immediately with feed delivery into the water. Vulnerablebioactive agents started to leach and decompose when the feed becomesoaked with water, and potentially harming the surrounding endogenousorganisms in the ecosystem.

To overcome some of the disadvantages associated with the delivery indry pelleted feeds, the active agent has been encapsulated withinmicrocapsules. Several types of natural or synthetic polymers have beenproposed for use as a matrix for binding and the controlled release ofactive agents. Examples of such polymers are poly(vinylpyrrolidone),poly(vinylalcohol), poly(ethylene oxide), cellulose and its derivates,silicone and poly(hydroxyethylmethacrylate). Biodegradable matrices areof interest since the degradation of natural polymers likepolysaccharides or starches occurs naturally in the aquatic environment.U.S. Pat. No. 4,239,754 describes a system where a nutritional componentsuch as free amino acids, and hormones are entrapped in a liposome andthe liposome is further encapsulated in a hydrocolloid matrix. Theresulting lipogel microcapsules were either stored as a freeze-driedpowder or suspended in water. This type of liposomal membrane or barrieris fragile, potentially expensive and difficult to make and would notlikely remain a discrete microcapsule when combined with othermaterials, or act as an appropriate part of a desirable aquatic invasivespecies management program.

The encapsulating polymers described in the art do not solve all of theproblems associated with delivering the active agent in the aquaticenvironment. Production of active agents in liposomes and theirsubsequent encapsulation in a hydrocolloid matrix is a labor-intensiveprocess that adds to the cost of the final product. Drying themicroencapsulated active results in oxidation and deactivation of theactive component, and more significantly renders the active agentinsoluble and thus not bio-available by the organism. Micro-encapsulatedactives that are stored in a dry state still have some of the samedisadvantages as described for dry pelleted feeds, as they must still berehydrated and manually introduced into an aquatic environment. Further,the microencapsulating polymers described in the prior art have noteliminated the decomposition and water leaching problems associated withthe use in aquatic environments.

The principle utility of the composition of the present invention lieswith its unique controlled buoyancy and bioadhesive matrix, in which theactive agent is dispersed in a form of oily droplets. The oil dispersedactive agent is enclosed within a particle matrix and will not leacheven after extended exposure in water. The bioadhesive polymeric matrixremains intact in the water body wherein mucosal tissues such as gill,skin and digestive tract of the targeted aquatic organism are exploitedfor uptake and release of the active agent. The method of producing anddelivering the composition is economical, environmentally safe andapplicable to both freshwater and marine waters. Use of the invention isparticularly attractive in controlling major invasive species such asfish, mussel and clam.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a biodegradable and bio-adhesivecomposition that binds or adheres to mucosal tissues and releases abioactive agent upon consumption by the aquatic organism and methods formaking and targeting delivery of such a composition to an aquaticorganism.

In some aspects, the invention provides a biodegradable and bio-adhesivepolymer composition, wherein said composition includes natural orsynthetic biodegradable polymers and wherein said polymers arebiopolymers or modified biopolymers of nucleic acids, amino acids, fattyacids and/or sugar monomers and wherein the synthetic polymers areplastics and/or elastomers.

In some aspects, the invention provides a bio-adhesive polymercomposition, as above, wherein said polymer includes poly-cationic orpositively charged polymers, such as chitosan and modified chitosan,poly-lysine, poly-ethylenimines (PEI), cationic agar, cationic plasticor latex and polymerizable cationic surfactants and the like.

In some aspects, the invention provides a biodegradable polymericcomposition in the form of a dry or wet particulate, macroparticle or amicro-particle wherein an active compound is embedded within theparticle polymeric matrix.

In some aspects, the invention provides a composition having a densitythat is adjustable to achieve neutral or controlled buoyancy in variousaquatic environments. In some aspects, the invention provides acomposition that remains intact for a desirable period of time uponexposure in water and wherein the embedded active compound is dissolvedor dispersed in an organic solvent and will not leach in water. In someaspects, the invention provides a composition wherein the embeddedbioactive compound released from the composition upon consumption by theaquatic organism. In some aspects, the invention provides a method formaking a biodegradable composition having bioadhesive and adjustabledensity properties including; Forming a slurry containing apoly-cationic polymer and buoyancy regulating materials; dissolving anactive compound in a mixture of water insoluble organic solvents andcationic surfactants; mixing the dissolved active compound in thebio-adhesive polymer slurry; adding attractant nutrient such as fishmeal, a protein, a lipid, or a resistant starch to the slurry;pelleting, granulating or atomizing the slurry and hardening theparticulate slurry or microdroplets to form solid wet or dry particlesin a desirable shape and size distribution.

In some aspects, the invention provides a method for delivering abioactive agent such as a toxicant, therapeutic or nutraceuticalcompounds to an organism in aquatic ecosystems including; producingbioadhesive composition containing a bioactive agent; adjusting the sizeand density of said composition to maximize its availability to theaquatic organism in the targeted water body; dispersing the bioactiveparticulated composition in an aquatic environment at a sufficientamount to obtain a desired effect on the aquatic organism.

In some aspects of the invention, the bioactive agent is an aquaticbiocide or a biological control agent, wherein the agent controlsvarious undesirable vertebrate and invertebrate aquatic organisms suchas fish, mussel, clam, sea snail and the like.

In some aspects of the invention, the bioactive agent is a therapeuticagent such as a bactericidal antibiotic or peptide, wherein thetherapeutic compound is capable of treating diseased farmed fish.

In some aspects of the invention, the bioactive agent is a nutraceuticalagent such as essential fatty acids, carotenes, amino acids, proteins,peptides, hormones and vitamins.

In some aspects of the invention, the polymeric composition containsmatrix forming polymers such as alginate, pectin, gelatin, agar,carrageenan, or their modified polymers and a mixture thereof, whereinthe bioadhesive polymer such as chitosan, cationic guar and/or otherpoly-cationic polysaccharides is admixed in.

In some aspects of the invention, the polymeric composition containsmatrix forming polymers wherein the matrix forming polymer is thebioadhesive polymer itself such as chitosan, cationic guar and/or otherpoly-cationic polysaccharides.

In some aspects of the invention, the density of the composition isadjusted by incorporating, in various proportions, water insoluble metalsalts or minerals and molten fats, waxes and/or polypropylene wax.

In some aspects of the invention, the composition is pelleted,particulated or atomized into a desired shape and size and hardened bycross-linking salts or chemicals.

In some aspects of the invention, the pelleted or particulatedcomposition is delivered wet or dried in any known drying methodincluding air or vacuum drying and delivered as dry pellets orparticulated powder.

In some aspects the invention provides a bio-adhesive composition forreleasing an active compound in the digestive tract of an aquaticorganism.

In some aspects the invention provides a bio-adhesive compositioncontaining a biocide or a mixture of biocides in the form of wet or drymicro-particle in various size ranges depending on the size preferenceof the targeted invasive organism. For example, the particle size may beabout 5-10 microns for eradicating invasive mussels, such as Zebramussels, or about 50-150 microns for eradicating invasive fish species,such as Asian carp.

These and other aspects of the present invention will become apparentfrom the following specification.

BRIEF DESCRIPTION OF THE DRAWINGS

It is to be understood that the disclosed drawings are merely exemplaryrepresentative of the invention that may be embodied in various forms.Therefore, specific functional details disclosed herein are not to beinterpreted as limiting, but merely as a basis for the claims and as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention.

FIG. 1 is a light microscopic view of density-adjusted microparticlescontaining oil droplets embedded in a matrix of bio-adhesive polymer ofthe presently claimed invention. The upper picture shows microparticleshaving their density adjusted for fresh water application, and the lowerpicture shows microparticles having their density adjusted for marinewater application.

FIG. 2 is a view of density-adjusted pellets containing oil dropletsembedded in a matrix of alginic acid polymer and bio-adhesive polymer ofthe presently claimed invention. The left picture shows dry pellets andthe right picture shows wet pellets having their density adjusted forsinking in marine water environment.

FIG. 3 demonstrates the controlled buoyancy property (sinking rate inmm/sec) of the composition of the present invention in various waterbodies as a function of size and density.

FIG. 4 shows the effect of particle size and density on the sinking rate(mm/sec) in fresh water simulated conditions.

FIG. 5 illustrates that the settling velocity (V_(s)) of the compositionof the present invention conforms to Stokes law as depicted from thefollowing equation:

V _(s)=( 2/9)((ρρ−ρf)/μ)(g*R ²)

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the following description of the invention, it is to be understoodthat the terms used have their ordinary and accustomed meanings in theart, unless otherwise specified. The term “active agent,” “bioactivecompound,” “Biological control agent,” is intended to broadly refer toany toxic, therapeutic or nutraceutical substances capable of treatingdifferent forms of living organisms used in fields such as aquaticecosystems, agriculture and aquaculture. A “Toxic Substance” as definedby the U.S. Environmental Protection Agency (EPA) is “any substance ormixture of substances intended for preventing, destroying, repelling, ormitigating any pest.” A toxic agent may be a chemical substance orbiological agent (such as a virus or bacteria) used against pestsincluding aquatic invasive organisms, which includes pesticides,piscicides, fungicides, herbicides, insecticides, algaecides andmoluscicides. Selective pesticides kill a specific target organism whileleaving the desired species relatively unharmed. Nonselective pesticideskill all species with which they come into contact. Suitable aquaticbiocides according to the present invention are those registered andregulated by the EPA such as Antimycin A, Piperonyl Butoxide (PBO),Pyrethrins, Rotenone and Cube Resins other than Rotenone, Niclosamide,aminoethanol salt (such as Bayluscide), Trifluoromethyl-4-nitrophenol(TFM) and the like. A therapeutic substance tends to overcome diseaseand promote recovery and includes any antimicrobial substance or drugsuch as a germicide, antibiotic, antibacterial, antiviral, antifungal,antiprotozoal, antiparasitic and therapeutic proteins and peptides. Anutraceutical substance tends to provide health and medical benefits andincludes essential fatty acids such as DHA, EPA and ARA, essential aminoacids such as lysine, methionine, arginine and the like, vitamins suchas vitamin A, C, D, E and the like, proteins and peptides.

The terms “Aquatic Non-indigenous organism” and “Aquatic Invasiveorganism or species,” are intended to broadly refer to any aquaticorganisms that have been introduced into new fresh water or marineecosystems and are both harming the natural resources in theseecosystems and threatening the human use of these resources. Aquaticinvasive organisms according to the present invention would include anyspecies of fish, shellfish, mussel, mollusks, clam and jellyfish.

The term “Biodegradable polymer,” is intended to broadly refer to anypolymer susceptible to degradation by biological activity, with thedegradation accompanied by a lowering of its molar mass.

The term “Cationic or poly-cationic polysaccharide” is intended tobroadly refer to any naturally occurring or modified or syntheticcationic polysaccharides, as well as polysaccharides and modifiedpolysaccharide derivatives that have been made cationic by chemicalmeans. This includes, for example, quarternization with variousquaternary amine compounds containing reactive chloride or epoxidesites.

The term “Bioadhesive or mucoadhesive polymer” is intended to broadlyrefer to any suitable cationic polymer that readily bind to negativelycharged tissues or organs such as gill or gastric mucosal tissues andtransporting bioactive material across cell membranes. Examples ofcationic polysaccharides include, but not restricted to, cationichydroxyethyl cellulose and cationic hydrophobically modifiedhydroxyethyl cellulose, linear macromolecules such as polyethyleneimine(such as Lupasol® by BASF), poly-L-lysine (PLL) or other poly-cationicamino acids, Chitosan, modified chitosans such as dimethyl, trimethyland carboxymethyl chitosan, cationic guar and/or other poly-cationicpolysaccharides, diethylaminoethyl-dextran (DEAE-dextran), and branchedpolymers such as poly (amidoamine) (PAMAM) dendrimers and POLECTRON® 430(by International Specialty Products).

The biodegradable polymeric matrix composition of the inventioncomprises a polymer susceptible to degradation by biological activity inthe aquatic ecosystem. In the broadest aspects of the invention, anynatural or synthetic polymer is contemplated to be suitable, includingbut not limited to, starches and modified cellulose such as ethyl,methyl and carboxymethyl-cellulose and the like; polysaccharides andgums such as agar, carrageenan, alginate, pectin, Chitosan, modifiedChitosan, guar gum and the like; proteins such as gelatin, milkproteins, glutens, soy and pea protein isolates, Zein and the like; andmolten fats such as saturated or hydrogenated fats, waxes, fatty acidalcohols of longer than 12 carbon chain and paraffin oils.Biodegradation of synthetic polymers can be accomplished by synthesizingthe polymers with hydrolytically unstable linkages in the backbone,which is commonly achieved by the use of chemical functional groups suchas esters, anhydrides, orthoesters and amides. Most commonly usedbiodegradable synthetic polymers are poly(glycolic acid) (PGA),poly(lactic acid) (PLA) poly(acrylic acid or methacrylates) and theircopolymers, as well as other materials, including poly(dioxanone),poly(trimethylene carbonate) copolymers, and poly(e-caprolactone)homopolymers, polyvinyl pyrrolidone, derivatives of polyvinylpyrrolidone and copolymers of such.

Generally, a matrix polymer that remains intact in the aquaticenvironment in the form of a particle for at least several hours ispreferred. The backbone polymers of the matrix may essentially be anyhydrophilic polymer and preferably such polymers that may be suitablefor cross-linking. The preferred matrix polymer is selected from thegroup consisting of hydrogel polymers and combinations thereof,preferably but not necessarily, cross-linked hydrogel polymers such asalginate, pectin, chitosan, agar, cationic agar, carrageenan, gelatinand combinations thereof. The matrix polymer is preferably used in anamount of between 0.01 and 20% by weight with respect to the totalweight of the composition. More preferably, this amount is between 0.05and 15% by weight with respect to the total weight of the compositionand more preferably between 1 and 10% by weight.

The biodegradable polymeric matrix composition of the inventioncomprises a bioadhesive or mucoadhesive polymer as a delivery vehiclefor a bioactive agent. In the broadest aspects of the invention, anycationic or positively charged polymer is contemplated to be suitable,including but not limited to, cationic hydroxyethyl cellulose andcationic hydrophobically modified hydroxyethyl cellulose,polyethyleneimine, diethylaminoethyl-dextran, poly-L-lysine (PLL),chitosan, modified chitosans such as dimethyl, trimethyl andcarboxymethyl chitosan, cationic guar and/or other poly-cationicpolysaccharides. In a more preferred aspect of the invention, anychitosan and/or modified chitosan are suitable.

In one embodiment of the present invention, the bioadhesive polymer isalso serving as the matrix polymer wherein the active agent is embeddedin the matrix.

In another embodiment of the present invention, the bioadhesive polymeris added to the matrix polymer to provide adhesive properties to thematrix. The bioadhesive polymer is preferably used in an amount ofbetween 0.01 and 20% by weight with respect to the total weight of thecomposition. More preferably, this amount is between 0.05 and 15% byweight with respect to the total weight of the composition, and mostpreferably, between 1 and 10% by weight.

The biodegradable polymeric matrix composition of the inventioncomprises a mixture of metals or water insoluble salts and natural orsynthetic molten fats or waxes in a specific desirable ratio to achieverequired particle buoyancy in the targeted aquatic ecosystem. In thebroadest aspects of the invention, any water insoluble salt having adensity greater than 1 g/cm⁻³, including but not limited to carbonates(CO₃ ²⁻), phosphates (PO₄ ²⁻) and sulfates (SO₄ ²⁻) of Ag, Ba, Ca, Mgand Zn and the like, and any molten fats and natural or synthetic waxeshaving a density lower than 1 g/cm⁻³, including but not limited to thosefrom plants, insects and animals source, and synthetic waxes that areprimarily derived by polymerizing ethylene or alpha olefins arecontemplated to be suitable. In a more preferred embodiment of theinvention, a mixture of tricalcium phosphate (TCP) and polypropylene wax(PPP) is suitable. The preferred amount of TCP and PPP mixture isbetween 0.01 and 40% by weight with respect to the total weight of thecomposition. More preferably, this amount is between 0.05 and 30% byweight with respect to the total weight of the composition. Thepreferred ratio between the TCP and the PPP is any desirable ratio thatis required to achieve the specific particle buoyancy in the targetedaquatic ecosystem. For example, it is to be understood that acomposition suitable for treating fresh water ecosystems would containlower TCP/PPP ratios than a composition suitable for treating marinewater ecosystems.

The active compound in the context of the present invention is abiocide, a therapeutic or a nutraceutic substance. A biocide substanceis a chemical or a biological control agent, capable of killing,preventing, destroying, repelling, or mitigating various undesirableaquatic organisms such as fish, mussels, clams and the like. In thebroadest aspects of the invention, any type of biocide or biologicalagent (such as a dry toxic virus or bacteria) may be used, including butnot limited to pesticides, piscicides, fungicides, herbicides,insecticides, algaecides, moluscicides, and the like. Preferred aquaticbiocides according to the present invention are those registered andregulated by the U.S. Environmental Protection Agency (EPA) such asAntimycin A, Piperonyl Butoxide (PBO), Pyrethrins, Rotenone and CubeResins other than Rotenone, Niclosamide, Aminoethanol salt (such asBayluscide product produced by BASF), Trifluoromethyl-4-nitrophenol(TFM) and the like.

In one embodiment of the present invention, the bioactive substance is atherapeutic agent such as a bactericidal peptide or antibiotic thatkills infectious bacteria in targeted aquatic ecosystems or inaquaculture systems. In the broadest aspects of the invention, any typeof an antibiotic may be used, including but not limited to germicides,antibacterials, antivirals, antifungals, antiprotozoals andantiparasitics. Specific antibiotic substances includes sulfamides,diaminopyrimidines, penicillins, tetracyclines, cephalosporins,aminoglucosides, chloramphenicol and derivatives, quinolones andfluoroquinolones, nitrofurans, nitroimidazoles and mixtures thereof. Inanother embodiment of the present invention, the bioactive substance isa nutraceutical compound such as a protein, a peptide, an oil, avitamin, or a hormone. In the broadest aspects of the invention, anytype of a nutraceutical may be used, including but not limited toessential fatty acids such as DHA, EPA and ARA and the like, essentialamino acids such as lysine, methionine, arginine and the like, vitaminssuch as vitamins A, C, D, E and the like, carotenes such as betacarotene, leutene, astaxanthin and the like.

Generally, the bioactive agent is solubilized in an organic solventbefore embedding in the polymeric matrix of the present invention. Inthe broadest aspects of the invention, any type of oil, fat or greasecan be used for solubilizing including but not limited to naturallyoccurring or synthetic oils in either liquid or solid form at ambienttemperature. Suitable oil, in the context of the present invention,encompasses all kinds of oil bodies or oil components, in particular,fish oil, vegetable oils like rape seed oil, sunflower oil, soy oil,olive oil, cocoa butter, coconut oil, palm oil, castor oil, and thelike, modified vegetable oils like alkoxylated sunflower or soy oil,synthetic (tri) glycerides like technical mixtures of mono, di andtriglycerides of C6-C22 fatty acids, and fatty acid alkyl esters likemethyl or ethyl esters of vegetable oils. Preferably, the solubility ofthe bioactive agent in oil is enhanced by the use of surface-activeagents such as cationic or nonionic surfactants and most preferablycationic surfactants. Typical non-limiting examples for cationicsurfactants are quaternary ammonium salts such astrimethylalkylammonium, chlorides or bromides of benzalkonium andalkylpyridinium ions and amines with amide linkages, polyoxyethylenealkyl and alicyclic amines and the like. The preferred amount of thebioactive agent in the oil is between 0.01 and 10% by weight. Morepreferably, this amount is the maximal attained solubility of the agentin a given oil/surfactant system. The bioactive/oil solution ispreferably used in an amount of between 0.1 and 40% by weight withrespect to the total weight of the composition. More preferably, thisamount is between 0.5 and 30% by weight with respect to the total weightof the composition and more preferably between 5 and 30% by weight.

In one embodiment of the present invention, the bioactive and oilsolution is coated onto carrier particles of a nutrient such as aprotein, lipid or starch. In the context of the present invention, thesenutrients also serve as nutritional attractants for the targeted aquaticorganism to actively consume the composition from the aquatic medium.

In another embodiment of the present invention, the nutrient ispreferably taken-up and digested by the targeted aquatic organism. Forexample, it has been shown that bivalve mussels can, due to their largegill surface areas and the great amounts of water pumped through theirmantle cavity, successfully compete with other invertebrates in uptakeof certain organic matter such as free amino acids [12, 13]. Theinclusion of nutrients in the polymer matrix also provides sites andpores opened by the organism digestive enzymes, which allows thebioactive to be taken up more efficiently. In the broadest aspects ofthe invention, any type of nutrient can be used including but notlimited to amino acids, peptides, enzymes, proteins, protein isolatesand meals either from animal or plant source, fatty acids, lipids andstarches and their derivatives. Suitable nutrients, in the context ofthe present invention, encompass all kinds of meals, proteins and freeamino acids and starch or a mixture thereof, in particular fishmeal orprotein isolates from animal or plant source. The preferred amount ofthe nutrient in the composition of the invention is between 0.1 and 70%by weight, with respect to the total weight of the composition. Morepreferably, this amount is between 5 and 60% by weight with respect tothe total weight of the composition and more preferably between 10 and50% by weight.

In accordance with the objectives of the invention, the biodegradableand bioadhesive composition delivery vehicle for an aquatic organism ismade, dry or wet, and has a particle size in the range of from about 2microns to about 10,000 microns. The delivery vehicle is made from acomplex of components as disclosed above, including any type ofbiodegradable polymers such as soluble and resistant starch, gums suchas agar, pectin, carrageenan, ethyl, methyl or carboxymethyl cellulose,alginate, wax, fat or protein and a mixture thereof. The gel matrix ofthe particle is formed by hardening or cross-linking the polymers toprovide a stable and intact particle in the aquatic environment. Theprovided particles are attractive and ingestible by the aquaticorganism.

In one embodiment of the preparation method, a solution containing0.01-10% matrix forming polymer such as alginate or pectin is prepared.A solution containing 0.01-10% bioadhesive polymer such ashydrophobically modified hydroxyethyl cellulose, polyethyleneimine,diethylaminoethyl-dextran, poly-L-lysine (PLL) or chitosan is preparedseparately and homogenized with the polymer matrix solution. Buoyancycontrol compounds containing a mixture of metal salt and hydrophobic waxsuch as a mixture of TCP and PPP in a desirable ratio is added in anamount between 0.01 and 20% by weight of the polymer solution andhomogenized until a smooth slurry is obtained. About 0.1-2% of anemulsifier such as monoglycerides, sorbitan esters, propylene glycolesters, lecithin, polysorbates and sucrose esters of medium and longchain saturated fatty acids can be added to assist with the dispersionof the hydrophobic wax in the slurry.

In an alternative embodiment, the bioadhesive polymer is used to alsoform the matrix solution, such as a chitosan or cationic agar solution.In one more alternative embodiment, the particle matrix is formed withnegatively charged polymers, such as an alginate or pectin followed by abrief soaking of the preformed micro particles in a solution containingpositively charged polymer, such as chitosan, PLL or cationic agar, andthe like.

In one preferred embodiment, the bioactive substance is solubilized in amixture of an organic solvent and a cationic surfactant. The preferredorganic solvent is any vegetable or animal oil, preferably short carbonchain oils such as oils containing caprylic, capric or lauric acid. Mostpreferred short carbon chain oil is castor oil. The preferred cationicsurfactant is a quaternary ammonium salts surfactant. The bioactivesolution is prepared by dissolving the maximal soluble amount of thebioactive in the oil and surfactant system. The bioactive and oilsolution can be directly homogenized in the polymer matrix solution inan amount from about 0.01 to about 20% by weight of the polymer solutionor coated first on fine particles of a nutrient such as fish meal or soyor pea protein isolate in a ratio of 0.5-1:1 of bioactive and oilmixture: nutrient and then add-mixing the coated nutrient into thepolymer matrix solution.

The final slurry is hardened by cross linking or cooling after forming agel pellet in a desirable size and shape or is atomized using an air,ultrasonic or rotary atomizer, or any other atomizing means known in theart, into a water solution containing 0.1-10% cross-linking compoundssuch as calcium chloride for cross linking polymers such as pectin oralginate, potassium citrate for cross linking carrageenan based polymersor tripolyphosphate for cross linking chitosan polymers and the likes.The hardened matrix particles are then collected from the cross linkingsolution and can be packaged wet for application to the aquaticenvironment in their wet form, or dried by any means known in the artsuch as air drying, fluidized-bed drying, freeze/vacuum drying, and thelike and packaged dry for later use. In an alternative embodiment, theslurry can be spray dried into hot air chamber and the dry particlescollected and stored for later use.

In one embodiment of the present invention, the bioactive composition ina desirable size range and density is applied in any fresh, brackish ormarine aquatic ecosystem so that, upon contact with water, saidcomposition remains intact in the water for at least several hours andthe bioactive is retained within the composition. The composition isthen actively consumed and adheres to mucosal tissues of the targetedorganism wherein the bioactive compound is released and absorbed by theaquatic organism. In another embodiment, a composition containing abiocide or a mixture of biocides is target-delivered to an undesirableaquatic organism. For example a 1:1 mixture of Rotenone and piperonylbutoxide (PBO) is utilized in the composition of the present invention,in a size range between 5 and 10 microns. The density of themicroparticle is adjusted with an appropriate TCP/PPP ratio to slightlyabove the fresh water density and fresh water bodies are treated withthe composition to impede habitats of mussels and sea lamprey species.

In yet another embodiment of the present invention, antimycin A isutilized in the composition of the present invention, and microparticlesin a size range between 50 and 100 microns are formed. The density ofthe microparticles is adjusted with an appropriate TCP and PPP ratio tomatch the density of aquatic estuaries for control of detrimental orinvasive aquatic animal species, such as Asian carp or crayfish.

In still another embodiment of the present invention, the antibioticoxytetracycline is utilized in the composition of the present invention,and pellets in a size range between 2000 and 5000 microns are formed.The density of the pellets is adjusted with an appropriate TCP/PPP ratioto match the water density of a marine environment, and the pellets usedto treat aquatic farmed species, such as a salmonid, against a bacterialinfection.

In further embodiment of the present invention, astaxanthincontaining—microbial cells or oil extract is utilized in the compositionof the present invention, and pellets in a size range between 5000 and10000 microns are formed. The density of the pellets is adjusted with anappropriate TCP/PPP ratio to match the water density of a marineenvironment, and the pellets fed to salmon fish about 30 days beforeharvesting to provide a desirable pigmentation in the fish flesh.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Production of Bioadhesive Composition ContainingRotenone for Fresh Water Application

In a 40 L stainless steel vessel, 16 L of distilled water was added.Sodium alginate (about 200 g, Manugel® DMB, FMC Biopolymer,Philadelphia, Pa.) was slowly added to the distilled water in thestainless steel tank under a vigorous mixing (2,000 RPM, RS-02, Admix,Manchester, N.H.) until completely dissolved. Liquid soy lecithin (about120 g, Archer-Daniels-Midland Co., Decatur, Ill.) and Tween 80 (about120 g, Sigma) were added to the alginate solution and the solutioncontinued to emulsify for 15 minutes under vigorous mixing (2,000 RPM).Polypropylene wax (about 1000 g, Propylmatte-31, Micro Powders, Inc.,Tarrytown, N.Y.) was added to the alginate solution and the solutioncontinued to emulsify for additional 15 minutes under vigorous mixing.The pH of the slurry was then adjusted to 6.2 with 1M glacial aceticacid.

In a separate small 10 L stainless steel vessel, 4 L of distilled waterwas added and warmed to 50° C. Chitosan (about 120 g, high viscositychitosan 90% DA, MayPro, Purchase, N.Y.) was slowly added to the warmedwater. Chitosan, which is the structural element in the exoskeleton ofcrustaceans (crabs, shrimp, etc.) is bioadhesive and readily binds tonegatively charged entities. It is a linear cationic polysaccharidecomposed of randomly distributed β-(1-4)-linked D-glucosamine(deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).Chitosan, is produced commercially by deacetylation of chitin (can beproduced from chitin also). The degree of deacetylation (% DA) incommercial chitosans is in the range 60-100%. One hundred fifty (150) gof glacial acetic acid was carefully added (with mixing) to the warmwater under a vigorous mixing (2,000 RPM) until the chitosan completelydissolved. The chitosan solution was cooled to room temperature and thepH adjusted to 6.2 with 50% sodium hydroxide solution. The chitosansolution was then combined with the alginate solution under vigorousmixing.

In a one (1) L glass beaker in a fume hood, 20 g of Rotenone (analyticalgrade, Sigma) was added and dissolved in an equal amount of chloroform.Optionally, the toxicity of the Rotenone can be further enhanced byadding 20 g piperonyl butoxide (PBO, Sigma) with the Rotenone. About twohundred (200) g castor oil (Sigma), 20 g cationic surfactant (CationicEmulsifier-1, Abitec Corp., Janesville, Wis.) and about 200 g olive oil(available from a local store) were added and mixed together to obtain aclear oily solution. The beaker containing the dissolved Rotenone wasplaced in 40° C. water bath in the hood and under a stream of nitrogenfor about two (2) hours to allow the chloroform to evaporate. Thedissolved Rotenone was then slowly mixed with about 1880 g soy proteinhydrolyzate (Solae™, Solae LLC, St. Louis, Mo.) until the powderappeared uniformly wet. The Rotenone coated soy protein then slowlyadded into the alginate/chitosan slurry and gently mixed (500-1000 RPM)until smooth slurry was obtained. Alternatively, the soy protein powdercan be mixed separately in the alginate/chitosan slurry followed bymixing in the oil solubilized Rotenone.

Microparticles formation: The Rotenone containing slurry was atomized(Air atomizer ¼ JAU-SS, Spraying Systems Co., Wheaton, Ill.) under 25psi air pressure and microparticles formed by cross linking in a waterbath containing 2% calcium chloride. Microparticles in a size rangebetween 50 microns and 150 microns were screen sieved were evenly spreadon a tray at a loading capacity of 1000 g/sq ft and placed on a shelf ina freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.). Vacuum pressurewas then applied at 100 mTORR and shelf temperature raised to +40° C.Drying was completed within 24 hours. Alternatively, the wetmicroparticles can be dried in a vacuum dryer or fluidized bed dryer.The composition of the microparticles is provided in Table 1, below.

TABLE 1 Rotenone microparticle composition (g dry weight/100 g) Alginate6 g Chitosan 3 g Soy lecithin 3 g Tween-80 3 g Propylmatte-31 26 g Soyprotein 48 g Castor oil 5 g Olive oil 5 g Cationic emulsifier 0.5 gRotenone 99% crystalline 0.5 g

FIG. 1 depicts a light microscope image of the mucoadhesivemicroparticles of the present invention having adjusted density forfresh water (upper picture) or marine water (lower picture)applications. The Rotenone or Rotenone/PBO microparticles are useful, inaccordance with local and federal regulations and registrationrequirements, for preventing both aquatic invertebrate and vertebrateinvasive organisms.

Example 2 Production of Bioadhesive Microparticles Containing Antimycina for Fresh Water Application

In a 20 L stainless steel vessel, 10 L of distilled water was added andwarmed to 50° C. Chitosan (about 300 g, high viscosity chitosan 90% DA,MayPro, Purchase, N.Y.) was slowly added to the warmed water. Threehundred (300) g of glacial acetic acid was carefully added (with mixing)to the warm water under a vigorous mixing (2,000 RPM) until the chitosancompletely dissolved. The chitosan solution was cooled to roomtemperature and the pH adjusted to 6.2 with 50% sodium hydroxidesolution. Liquid soy lecithin (about 60 g, Archer-Daniels-Midland Co.,Decatur, Ill.) and Tween 80 (about 60 g, Sigma) were added to thealginate solution and the solution continued to emulsify for 15 minutesunder vigorous mixing (2,000 RPM). Polypropylene wax (about 500 g,Propylmatte-31, Micro Powders, Inc., Tarrytown, N.Y.) was added to thealginate solution, prepared according to method described under Example1, and the slurry continued to emulsify for additional 15 minutes undervigorous mixing.

In 1 L glass beaker in a fume hood, 10 g of antimycin A (analyticalgrade, Sigma) was added and dissolved in an equal amount of chloroform.About one hundred (100) g castor oil (Sigma), 10 g cationic surfactant(Cationic Emulsifier-1, Abitec Corp., Janesville, Wis.) and about 100 gfish oil (available from a local vitamin store) were added and mixedtogether to obtain a clear oily solution. The beaker containing thedissolved antimycin A was placed in 40° C. water bath in the hood andunder a stream of nitrogen for about two (2) hours to allow thechloroform to evaporate. The dissolved antimycin A was then slowly mixedwith about 940 g soy protein hydrolyzate (Solae™, Solae LLC, St. Louis,Mo.) until the powder appeared uniformly wet. The antimycin A coated soyprotein then slowly added in the chitosan slurry and gently mixed(500-1000 RPM) until a smooth slurry was obtained. Alternatively, thesoy protein powder can be mixed separately in the chitosan slurryfollowed by mixing in the oil solubilized antimycin A.

Microparticles formation: The antimycin A containing slurry was slowlypoured on a rotating spinning disc (Southwest Research Institute(SwRI®), San Antonio, Tex.) to form narrow size distribution ofmicrodroplets between 50 microns and 100 microns. Hardened matrixmicroparticles were formed by cross-linking the chitosan polymers in awater bath containing 10% iso-propanol (70% purity) and 10%tripolyphosphate. Microparticles were harvested from the cross linkingbath after a hardening period of about two (2) hours and dried in afluidized bed dryer (Fluid Bed System model 0002, Fluid Air, Aurora,Ill.). The composition of the microparticles is provided in Table 2. Theantimycin A micro particles are useful, in accordance with local andfederal regulations and registration requirements, to restorethreatened/endangered fish to their native habitat (selective kill) andfor disease treatment of farmed fish.

TABLE 2 Antimycin A microparticle composition (g dry weight/100 g)Chitosan 14 g Soy lecithin 3 g Tween-80 3 g Propylmatte-31 25 g Soyprotein 44 g Castor oil 5 g Fish oil 5 g Cationic emulsifier 0.5 gAntimycin A 0.5 g

Example 3 Production of Bioadhesive Microparticles Containing anAntibiotic for Treating Infected Farmed Fish

In a 40 L stainless steel vessel, 16 L of distilled water was added.Sodium alginate (about 200 g, Manugel DMB, FMC Biopolymer, Philadelphia,Pa.) was slowly added to the distilled water in the stainless steel tankunder a vigorous mixing (2,000 RPM, RS-02, Admix, Manchester, N.H.)until completely dissolved. Liquid soy lecithin (about 120 g,Archer-Daniels-Midland Co., Decatur, Ill.) and Tween 80 (about 120 g,Sigma) were added to the alginate solution and the solution continued toemulsify for 15 minutes under vigorous mixing (2,000 RPM). A buoyancycontrol mixture of tricalciumphosphate (TCP, technical grade) andPolypropylene wax (TPP, Propylmatte-31, Micro Powders, Inc., Tarrytown,N.Y.) containing 330 g TCP and 260 g TPP was added to the alginatesolution and the slurry continued to emulsified for additional 15minutes under vigorous mixing. The pH of the slurry was then adjusted to6.2 with 1M glacial acetic acid.

In a separate small 10 L stainless steel vessel, 4 L of distilled waterwas added and warmed to 50° C. Chitosan (about 120 g, high viscositychitosan 90% DA, MayPro, Purchase, N.Y.) was slowly added to the warmedwater. One hundred fifty (150) g of glacial acetic acid was carefullyadded (with mixing) to the warm water containing chitosan under vigorousmixing (2,000 RPM) until the chitosan completely dissolved. The chitosansolution was cooled to room temperature and the pH adjusted to 6.2 with50% sodium hydroxide solution. The chitosan solution was then combinedwith the alginate slurry under vigorous mixing.

In 1 L glass beaker in a fume hood, 40 g of oxytetracycline (OTC, Sigma)was added and dissolved in an equal amount of 95% isopropyl alcohol.About two hundred (200) g castor oil (Sigma), 20 g cationic surfactant(Cationic Emulsifier-1, Abitec Corp., Janesville, Wis.) and about 600 gfish oil (available from a local vitamin store) were added and mixedtogether to obtain a clear oily solution. The dissolved OTC was thenslowly mixed with about 1800 g salmon protein isolate (MarineBioproducts AS, Norway) until the powder appeared uniformly wet. The OTCcoated salmon protein then slowly added to the alginate and chitosanslurry and gently mixed (500-1000 RPM) until smooth slurry was obtained.Alternatively, the salmon protein isolate can be mixed separately in thealginate and chitosan slurry followed by mixing in the oil solubilizedOTC.

Pellet formation: The OTC containing slurry was dropped and pellets wereform by cross linking in a water bath containing 2% calcium chloride.Pellets were collected and dried at 60° C. in a vacuum oven (Shel Lab,Cornelius, Oreg.). FIG. 2 depicts an image of the bioadhesive pellets ofthe present invention having adjusted density for marine water. The leftpicture shows freeze dried pellets and the right picture showsrehydrated pellets sinking in marine water. The composition of thepellets is provided in Table 3. The OTC pellets are useful for treatinga broad range of bacterial diseases in farmed fish.

TABLE 3 OTC microparticle composition (g dry weight/100 g) Alginate 6 gChitosan 3 g Soy lecithin 3 g Tween-80 3 g Propylmatte-31 7 gTricalciumphosphate 9 g Salmon proteins isolate 47.5 g Castor oil 5 gOlive oil 15 g Cationic emulsifier 0.5 g Oxytetracycline 1 g

Example 4 Production of Bioadhesive Microparticles Containing a Mixtureof Biocides for Treating Aquatic Infrastructure Such as Pipes, Pumps,Cables and Other Water Submerged Surfaces

Heavy sinking bioadhesive microparticles were produced as described inExample—2 with the exception of replacing the PPP with equal amount ofTCP and the soy protein isolate with equal amount of unmodifiedresistant starch (Hylon V, National Starch and Chemical, Bridgewater,N.J.). A broad mixture of biocides are prepared by mixing in a one (1) Lglass beaker in a fume hood, 10 g Rotenone, 10 g piperonyl butoxide(PBO) and 10 g antimycin A (all from Sigma) and dissolving them in anequal amount of chloroform. About two hundred (200) g castor oil(Sigma), 20 g cationic surfactant (Cationic Emulsifier-1, Abitec Corp.,Janesville, Wis.) and about 200 g coco-butter (available from a localstore) are added and mixed together in a water bath at 40° C. to obtaina clear oily solution. The beaker containing the dissolved biocides waskept in 40° C. water bath in the hood and under a stream of nitrogen forabout two (2) hours to allow the chloroform to evaporate. The dissolvedbiocides were then slowly mixed with about 1880 g resistant starch untilthe powder appeared uniformly wet. The biocides coated starch granulesare then slowly added to the chitosan solution and gently mixed(500-1000 RPM) until a smooth slurry is obtained. The solution is spraydried using 30″ spray dryer (S/S Mobile Minor, GEA Process EngineeringInc., Columbia, Md.). The dry micro particles, mostly in a size rangefrom 5 microns to 20, microns were collected and stored for later use.The heavy sinking microparticles are useful for treating water-submergedsurfaces such as pipes, pumps, cables and other submerged structuresagainst bottom feeder invasive organisms such as mussels and clams.

Example 5 Production of Bioadhesive Microparticles ContainingNiclosamide for Treating Aquatic Invasive Invertebrates

A 20 L Alginate and chitosan solution is prepared as described inExample 1, using a 1:3 mixture of TCP/PPP that provides slow sinkingmicroparticles. In a one (1) L glass beaker in a fume hood, 40 g ofniclosamide (Sigma) is added and dissolved in an equal amount ofacetone. About two hundred (200) g castor oil (Sigma), 20 g cationicsurfactant (Cationic Emulsifier-1, Abitec Corp., Janesville, Wis.) andabout 200 g coco butter (available from a local vitamin store) are addedand mixed in a water bath at 40° C. to obtain a clear oily solution. Thewarm oily solution is slowly mixed with about 1800 g resistant starch at40° C. until the powder appeared uniformly wet and then the mixture iscooled down to room temperature while mixing is continued. The powder iskept in the hood and under a stream of nitrogen for about two (2) hoursto allow the acetone to evaporate. The dissolved niclosamide is thenslowly added to the alginate/chitosan solution and gently mixed(500-1000 RPM) until a smooth and uniform slurry is obtained.

To form fine several microns size droplets, the slurry is slowly addedto another mixing vessel containing 50 L cold liquid paraffin orchloroform under vigorous homogenizing at 10,000 RPM. The emulsion iskept cold at below 10° C. to minimize potential leaching and loss ofniclosamide into the liquid paraffin. Two (2) L cold solution of 10%calcium chloride is slowly added to the emulsion under gentle mixing ofabout 500-1000 RPM and the preformed droplets allowed to cross-linkedand harden for about 30 min. The mixture is then allowed to settle andthe paraffin is discharged from the vessel. The wet intact niclosamidemicroparticles mostly in a size range between 4 microns and 12 micronsare stored for later use. Alternatively, the alginate/chitosan slurrycontaining the oil-dissolved niclosamide is extruded into cold (about10° C.) water bath containing 2% calcium chloride and the cross-linkedharden gel strings are harvested and dried in a convection oven, vacuumoven or freeze dryer and the like. The dried strings are then finelymilled to a particle size below 10 microns. The slow sinkingmicroparticles are useful against invasion of an invertebrate organismsuch as sea snail and sea lamprey.

Example 6 Density Adjustment of the Microparticles for Application inWater Bodies Having Various Salinities

Microparticles having various densities and size range were producedaccording to Example 1. The sink rate of the particles as a function oftheir size and water density is presented in FIG. 3. Low densitymicroparticles (having low TCP/PPP ratio) at an average particle size ofabout 100 micron remain in the upper one (1) meter depth of fresh waterbody for about 30 minutes and for about one (1) hour in marine waterbody.

Example 7 Sink Rate Adjustment of the Microparticles for Application ina Desired Water Body

Microparticles having various densities and size ranges were producedaccording to Example 1. The sinking rate of the particles as a functionof their size and density in fresh water body is presented in FIG. 4. Ithas been demonstrated that by varying the TCP/TPP ratio, according tothe claims of the present invention, the presence of 100 micronsmicroparticles in the upper one (1) meter depth of fresh water bodies isextended from about 30 minutes to about one (1) hour. FIG. 5demonstrates that the sinking rate of the microparticles in a givenwater body obeys Stokes law, thus allowing for the design of suchmicroparticles having a desirable sinking rate to target a specificorganism in the water body.

Example 8 Retention of Biocide Activity within the Composition DuringExposure in Water

Microparticles containing 10% dry weight olive oil were producedaccording to Example 1. The dry microparticles are placed in warm freshwater (40° C.) and the amount of oil leaching into the water wasmeasured over a time period of six (6) hours. Results showed that over80% of the oil remained within the microparticles even after 6 hoursexposure to in warm water. This example demonstrates the capacity of themicroparticles to retain commonly used biocides, which are mostly waterinsoluble, and prevent their exposure to non-targeted native organisms.While utilizing other functionalities of the microparticles such assize, sink rate and nutritional attraction to selectively target onlythe unwanted organism.

Example 9 Bio-Adhesive Properties of the Composition

Bio-adhesive microparticles excluding the bioactive and with or withoutchitosan are produced as described in Example 2. The bioadhesiveproperty of the microparticles is tested by adhering them with bacterialculture of Lactobacillus rhamnosus sp. Five hundred (500) mg of drymicroparticles in a size range between 100 microns and 150 microns areplaced on a small 50 micron mesh sieve. The particles are gently washedwith 100 ml of sterile PBS buffer followed by 100 ml of live bacterialculture containing 10E8 CFU/ml in PBS buffer. The microparticles thenwashed with 100 ml of sterile PBS buffer and transferred to a beakercontaining 100 ml sterile PBS buffer added with 1% Tween-80. Themicroparticle solution is homogenized at 10,000 RPM using a labhomogenizer and was serially diluted before plating on LMRS agar plates.The colony forming units (CFU) are recorded after 72 h incubation at 37°C. and calculated per mg dry weight particles. Results are presented inTable 4.

TABLE 4 Bio-adhesive properties of the microparticle Microparticleswithout chitosan 10E2 CFU/mg dry weight Microparticles with chitosan10E4 CFU/mg dry weight

This example shows the bioadhesive property of microparticle due to theincorporation of bioadhesive polymer such as chitosan within thealginate matrix. Thus, the microparticles can be administered as abio-adhesive device that adheres to mucosal tissue of the aquaticorganism (e.g. gill, skin, oral cavity and along the digestive system)for absorption of the biocidal active agent(s) through the organismmucosal tissue.

Example 10 Controlling the Over-Growth of an Invasive Organism Such asAsian Carp with Rotenone/PBO Microparticles

Dry microparticles containing a 1:1 mixture of Rotenone and PBO areproduced according to Example 1. An open water body located in arecreational river and which is overpopulated with Asian carp is dosedwith a quantity of the dry particles, so as to achieve a concentrationof 50 ppm as active biocides in the upper one (1) meter depth of thewater body. Biocide measurement shows Rotenone concentrations well above20 ppm for several hours, suggesting that most of the particles remainbuoyant in the water body, which allows for effective exposure of thebiocide to a large number of fish. Massive mortality and morbid fish isobserved the day following the treatment. Biocide measurement after 24hours shows Rotenone concentrations below 2 ppm, indicating that most ofthe particles disappeared from the upper 1 meter depth water body andwere either consumed by the fish or sunk to the bottom of the watercolumn where natural biodegradation of the particles can take place.Substantial cost savings are achieved due to the more efficient andselective application of the biocide microparticles.

Example 11 Treating Sick Trout Fish with OTC Microparticles

Trout broodstock are stocked at 10 kg per m³ of fresh water and attemperature of 10° C. Water quality is maintained by rapidly exchangingthe tank water through mechanical and biofiltration systems. Fish arefed 4 times daily a total ration of 1% body weight on a commercial feedand 0.5% (wet weight) microparticles as described in Example 3 for 7days. Blood samples were taken for OTC profile analysis and comparedwith fish fed only standard commercial feed containing 0.5% OTC. Resultsshow that fish more efficiently absorb OTC from the microparticles ofthe present invention than from the OTC containing feed.

Example 12 Pigmenting Salmon Flesh with Astaxanthin Pellets

Atlantic salmon fish are raised in open sea cages. Thirty (30) daysbefore harvest, the fish are fed 20% of their daily a total ration of 1%body weight on astaxanthin containing pellets as described in Example 3.Blood samples and flesh color are analyzed for astaxanthin content andcompared with fish fed standard commercial feed containing astaxanthin.Results show that fish more efficiently absorb astaxanthin from thepellets of the present invention than from the commercial astaxanthincontaining feed.

REFERENCES

The contents of all references cited herein are hereby incorporated byreference herein for all purposes.

U.S. Patent Documents

6,194,194 Molloy, Daniel Feb. 27, 2001 3,851,053 Cardarlleri, et al.Nov. 01, 1971 4,400,374 Cardarlleri, et al. Jul. 24, 1980 3,059,379Attoe, Osborne J. Oct. 23, 1962 4,428,457 Fikkers, et al. Sep. 24, 19824,019,890 Fujita, et al. Dec. 03, 1974 2,891,355 Nelson, Florian N. Jun.23, 1959 3,336,155 Rowe, Englebert L. Aug. 15, 1967 3,276,857 Stansbury,et al. Oct. 04, 1966 4,239,754 Sache, et al. Oct. 25, 1977

OTHER REFERENCES

[1-13]

-   1. Mack, R. N., D. Simberloff, W. M. Lonsdale, H. Evans, M. Clout,    and F. Bazzaz. 2000. Biotic invasions: causes, epidemiology, global    consequences, and control. Ecological Applications. 10(3): 689-710.-   2. Myers, J. H., Simberloff, D., Kuris, A. M., & Carey, J. R. 2000.    Eradication revisited—dealing with non-indigenous species. Trends in    Ecology and Evolution, 15, 316-320.-   3. Pimentel, D., L. Lach, et al. (2000). “Environmental and economic    costs of non-indigenous species in the United States.” Bioscience    50(1): 53-65.-   4. Varney, R. W. 2004. Fighting the Spread of Invasive Species in    Connecticut/Vermont. U.S. Environmental Protection Agency (EPA).    www.epa.gov/libraries/region1-   5. Joseph M. Caffrey, Stephanie Evers, Michael Millane and Helen    Moran. 2011. Current status of Ireland's newest invasive species—the    Asian clam Corbicula fluminea (Müller, 1774). Aquatic    Invasions (2011) Volume 6, Issue 3: 291-299.-   6. Aldridge D C, Elliott P, Moggridge G D (2006). Microencapsulated    BioBullets for the control of biofouling zebra mussels.    Environmental Scientific Technology 40: 975-979-   7. O'Neill, C. R. and MacNeill, D. B. 1991. The zebra mussel    (Dreissena polymorpha): an unwelcome north american invader. Coastal    Resources Fact Sheet, November 1991, Sea Grant, Cornell Cooperative    Extension, State University of New York.-   8. Kornis M S, Mercado-Silva N, Vander Zanden M J. 2012. Twenty    years of invasion: a review of round goby Neogobius melanostomus    biology, spread and ecological implications. J Fish Biol. 2012    February; 80(2):235-85.)-   9. Daniel P. Molloy and Denise A. Mayer, Overview of a Novel Green    Technology: Biological Control of Zebra and Quagga Mussels with    Pseudomonas fluorescens, Version 6: Updated Aug. 24, 2007.-   10. Claudi and Macke, 1994; Karen Perry J E John Lynn, Detecting    physiological and pesticide-induced apoptosis in early developmental    stages of invasive bivalves, Hydrobiologia (2009) 628:153-164-   11. D. F. Villamar and C. J. Langdon. 1993. Delivery of dietary    components to larval shrimp (Penaeus vannamei) by means of complex    microcapsules. Marine Biology, 115:635.-   12. D. Siebers and A. Winkler. 1984. Amino-acid uptake by mussels,    Mytilus edulis, from natural seawater in a flow-through system.    Helgoland Marine Search. Vol. 38, 189-199-   13. D.C. Sigee (Ed). Freshwater microbiology: biodiversity and    dynamic interactions of Microorganisms in the Aquatic Environment.    John Wiley and Sons, 2005-524 pages)

We claim:
 1. A composition for delivering a bioactive agent in anaquatic environment, comprising at least one bioactive agent, at leastone bioadhesive polymer, at least one density-adjusting compound and atleast one nutrient.
 2. The composition according to claim 1, wherein thebioactive agent is approximately 0.05% to 10% by weight, thebio-adhesive polymer is approximately 0.05% to 10% by weight, thedensity-adjusting compound is approximately 0.05% to 30% and thenutrient is approximately 0.05% to 50% by weight.
 3. The compositionaccording to claim 1, wherein the bioactive agent is chosen frombiocides, pharmaceutics and nutraceuticals and mixtures thereof andwherein the bioactive agent is dissolved in a mixture of oil andcationic surfactant.
 4. The composition according to claim 1, whereinthe bioactive agent is a biocide chosen from Antimycin A, PiperonylButoxide (PBO), Pyrethrins, Rotenone and Cube Resins other thanRotenone, Niclosamide, aminoethanol salt, Trifluoromethyl-4-nitrophenol(TFM) and mixtures thereof.
 5. The composition according to claim 1,wherein the bioactive agent is a pharmaceutic chosen from antibiotics,antibacterials, antivirals, antifungals, antiprotozoans, antiparasiticsand mixtures thereof.
 6. The composition according to claim 1, whereinthe bioactive agent comprises one or more antibiotics.
 7. Thecomposition according to claim 1, wherein the bioactive agent is anutraceutical agent chosen from proteins, peptides, fatty acids, aminoacids, vitamins, carotenes, hormones and mixtures thereof.
 8. Thecomposition according to claim 1, wherein the bio-adhesive polymer ischosen from the group consisting of cationic hydroxyethyl cellulose andcationic hydrophobically modified hydroxyethyl cellulose,polyethyleneimine, diethylaminoethyl-dextran, chitosan, modifiedchitosans such as dimethyl, trimethyl and carboxymethyl chitosan,cationic guar and mixtures thereof.
 9. The composition according toclaim 1, wherein the density-adjusting compound is chosen from the groupconsisting of water insoluble salts and natural and synthetic moltenfats or waxes and mixtures thereof.
 10. The composition according toclaim 1, wherein the density-adjusting compound is chosen from the groupconsisting of water insoluble carbonate (CO₃ ²⁻), phosphate (PO₄ ²⁻) andsulfate (SO₄ ²⁻) salts of Ag, Ba, Ca, Mg and Zn, molten fats, naturalwaxes, and synthetic waxes that are primarily derived by polymerizingethylene or alpha olefins and mixtures thereof.
 11. The compositionaccording to claim 1, wherein the nutrient is selected from the groupconsisting of animal or plant meals, proteins, fish protein isolate, soyprotein isolate, pea protein isolate, canola protein isolate, peptides,amino acids, fatty acids, animal or plant oils, starches, resistantstarches, modified starches and mixtures thereof.
 12. The compositionaccording to claim 1, wherein the bioactive agent is water insoluble andis dissolved in a mixture of castor oil and cationic surfactant.
 13. Thecomposition according to claim 1, wherein the bioadhesive polymer ismixed with a matrix-forming polymer selected from the group consistingof ethyl-, methyl- and carboxymethyl-cellulose, agar, carrageenan,alginate, pectin, gelatin, glutens, and molten fats such as saturated orhydrogenated fats, waxes, solid fatty acid alcohols and paraffin oilsand mixtures thereof.
 14. The composition according to claim 1, whereinthe density-adjusting compound is a mixture of tricalcium phosphate(TCP) and polypropylene wax (PPP) and wherein the ratio between the TCPand the PPP is any desirable ratio having percentage of TCP between 0and 100% depending on specific compound density required.
 15. Thecomposition according to claim 1, wherein the composition is in the formof wet or dry particles.
 16. The composition according to claim 15,wherein the particles remain intact in water and retain the bioactiveagent for at least two (2) hours.
 17. The composition according to claim15, wherein the particles have dimensions of approximately 5-50 microns.18. The composition according to claim 15, wherein the particles havedimensions of approximately 50-1000 microns.
 19. The compositionaccording to claim 15, wherein the particles have dimensions ofapproximately 1000-10,000 microns.
 20. A method of producing thecomposition according to claim 15, comprising steps of: (i) Preparing abioadhesive polymer solution; (ii) Forming a polymer slurry byemulsifying into the polymer solution a mixture of buoyancy adjustingcompounds in a ratio adapted to provide a predetermined desired particledensity according to Stokes Law; (iii) Dissolving a bioactive agent intoa mixture of oil and cationic surfactant; (iv) Coating the product ofstep (iii) onto a nutrient; (v) Mixing the coated nutrient in thepolymer slurry; (vi) Granulating or atomizing the slurry into particleshaving a desirable size and dimension; and (vii) Hardening the particlesthrough a physical or chemical reaction.
 21. The method according toclaim 20, wherein the bioadhesive polymer solution comprises a mixtureof alginate and chitosan.
 22. The method according to claim 20, whereinthe oil-dissolved bioactive agent and the nutrient are added separatelyinto the polymer slurry.
 23. The method according to claim 20, whereinthe particulate bioadhesive polymer slurry is hardened by air drying orcooling.
 24. The method according to claim 20, wherein the particulatebioadhesive polymer slurry is hardened by chemical reaction.
 25. Themethod according to claim 20, wherein the bioadhesive polymer slurry ishardened by dropping or atomizing into a water bath containingmultivalent cations or by changing the pH.
 26. A method of controllinginvasive organisms in an aquatic ecosystem, comprising dispersing, intoa water body requiring treatment, a composition comprising: (i)Approximately 0.05% to 10% by weight of at least one bioactive agentselected from the group consisting of Antimycin A, Piperonyl Butoxide(PBO), Pyrethrins, Rotenone and Cube Resins other than Rotenone,Niclosamide, aminoethanol salt (such as Bayluscide),trifluoromethyl-4-nitrophenol (TFM) and mixtures thereof; (ii)Approximately 0.05% to 10% by weight of at least one bioadhesive polymerselected from the group consisting of cationic hydroxyethyl celluloseand cationic hydrophobically modified hydroxyethyl cellulose,polyethyleneimine, diethylaminoethyl-dextran, poly-L-lysine (PLL),chitosan, modified chitosans such as dimethyl, trimethyl andcarboxymethyl chitosan, cationic guar and mixtures thereof; (iii)Approximately 0.05% to 30% by weight of at least one density-adjustingcompound selected from the group consisting of water insoluble salts,natural or synthetic molten fats or waxes and mixtures of any of these;and (iv) Approximately 0.05% to 70% by weight of at least one nutrientselected from the group consisting of animal or plant proteins,peptides, amino acids, fatty acids, animal or plant oils, starches andmodified starches and mixtures thereof.
 27. The method as recited inclaim 26, wherein the composition further comprises a matrix-formingpolymer mixed therein.
 28. The method as recited in claim 26, whereinthe composition is an intact microparticulate composition prepared by aprocess comprising atomizing a slurry to form microdroplets andhardening the microdroplets through a physical or chemical reaction. 29.The method as recited in claim 26, wherein the composition is an intactpelleted composition prepared by a process comprising pelleting a slurryand hardening the pellets through a physical or chemical reaction. 30.The method as recited in claim 26, wherein the composition is a drycomposition.